found that in a resistant house fly strain, a glutathione transferase is overtranscribed (Fournier et al., 1992). Resist- ance may also be qualitatively determined.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 267, No. 20, Issue of July 15, pp. 14270-14274,1992 Printed in U.S.A.
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Acetylcholinesterase TWO TYPES OF MODIFICATIONS CONFER RESISTANCE TO INSECTICIDE* (Received for publication, November 12, 1991)
Didier Fournier$, Jean-Marc Bride, Frederic Hoffmanng, and Franpois Karchs From the Centre de Recherche d’Antibes,Laboratoire de Biotogie des Invertkbres, F-06606, Antibes, France and the §Department of Zoology and Animal Biology, University of Geneva, 154 route de Malagmu, CH-1224 CEone-Boweries/ Geneva, Switzerland
Quantitative and qualitative changes in acetylcholinesterase confer resistance to insecticides. We have constructed several Drosophila melanogasterstrains producing various amounts of enzyme by P-mediated transformation. Toxicological analysis of these strains demonstrates that resistance to organophosphorus insecticides is correlated with the amount of acetylcholinesterase in the central nervous system. Resistance may also be qualitatively determined. Comparison of the Drosophila acetylcholinesterase gene between a resistant strain caught in the wild and a wild type susceptible strain only revealed onenucleotide transition resulting in the replacement of aphenylalanine by a tyrosine. Flies mutant for acetylcholinesterase and rescued with a minigene mutagenized for this same transition produced an altered enzyme which renders flies resistant to pesticides.
Although resistance of insects to insecticides is an important agricultural problem because it reduces the efficiency of treatments, this phenomenon provides a good model of adaptation of eukaryotes to a toxic environment. Resistance to insecticides results from three main mechanisms: 1) insecticide penetration is reduced, 2) the insecticide is more efficiently metabolized by esterases, mixed function oxidases, or glutathione transferases, and 3) the target of the insecticide is modified. Until now, genetic origins of the resistance to insecticides havereceived little attention at the molecular level. Gene amplification orovertranscription have been shown to be involved in someinstances. More than 250 copies of the gene coding for an esterase are present in some resistant insects (Mouchhs et al., 1986; Field et al., 1988). We recently found that in aresistant house fly strain,aglutathione transferase is overtranscribed (Fournier et al., 1992). Resistance may also be qualitatively determined. Indeed, sodium channels(targets of pyrethroids)and acetylcholinesterase (target of organophosphates and carbamates) show modified binding or catalytic properties in resistant strains (Smissaert, 1964; Pauron et al., 1989). Although pointmutationsare suspected to be involved in these qualitative modifications, they remain t o be demonstrated. * This work was supported by grants from the Institut National de la Recherche Agronomique (AIP“Cholinesterase”) and from the Swiss National Science foundation and the fonds Claraz (to F. K. and P. Spierer.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed INRA, Laboratoire de Biologie des InvertBbrks, Centre de Recherche d’Antibes, 06606, Antibes, France. Fax: 33-93-67-89-55.
Acetylcholinesterase (EC 3.1.1.7) terminates nerve impulse by catalyzing the hydrolysis of the neurotransmitter acetylcholine. It is a key enzyme in the insect nervous system in which the cholinergic system is essential. This property led to thedevelopment of inhibitors of this enzyme as insecticides. Organophosphates and carbamates are among the most commonly used; they covalently bind to theactive site (Aldridge, 1950) and cause the death of the insect. However, several insect strains escape poisoning because they possess an altered acetylcholinesterase which is less sensitive to theactive metabolite of the organophosphate. Among these species, a Drosophila strain (MH19), resistant to malathion has been isolated by Morton and Singh (1982). In Drosophila, genetic and molecular studies determined that acetylcholinesterase is specific for the central nervous system and is encoded by an unique locus, Ace. This gene has been cloned by chromosomal walking. A 4.3-kb’ message is translated into aprecursor which is processed and assembled in an unique protein. This protein is dimeric, glycosylated, and linked to the membrane via a glycolipid anchor. Several Ace- mutants have been isolated and successfully rescued by P element transformation using a minigene (Hall and Kankel, 1976; Hall and Spierer, 1986; Fournier et al., 1988; Hoffmann et al., 1992). Using P element transformation of wild type flies or Acemutants, we have constructed several strains producing various amounts of acetylcholinesterase. These transgenic strains enabled us to demonstrate thatthere is astrong correlation between the amount of acetylcholinesterase in the central nervous system and the susceptibility to insecticides. We also identified the point mutation responsible for the malathion resistance in the MH19 strain isolated by Morton and Singh (1982). In order to definitely prove that this mutation is responsible for the resistance, we have rescued Acemutants with a minigene carrying it. EXPERIMENTALPROCEDURES
Insect Toxicology-10 females were exposed to a paper soaked with 1 ml of insecticide solution in small Petri dishes. Mortality was recorded 16 h after.10 independent assays with four or five insecticide concentrations were performed for each strain. LDSO values were determined by fitting dose/mortality data tosigmoid curves. Evaluation of Acetylcholinesterase Amount-15 flies were ground individually in 500 pl of 10 mM Tris-HC1, 0.1% Triton X-100,1 M NaCl. Amounts of enzyme were estimated by recording activity in 100-p1aliquots by monitoring the hydrolysis of 1 mM acetylthiocholine at 25 “C (Ellman et al., 1961). One unit corresponds to the metabolization of 1 nmol of acetylthiocholine/min. Amounts of protein are related to activities when acetylcholinesterases are identical, i.e. without modification in their catalytic parameters. We thus used this method only for wild type enzymes.
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The abbreviation used is: kb, kilobase(s).
Acetylcholinesteraseand Resistance to Insecticide Evaluation of Catalytic Parameters-Reaction of irreversible cholinesterase inhibitors, such as organophosphorus compounds, is pseudo-first order with respect to inhibitor concentration.The following reaction scheme holds for the progressive inhibition of cholinesterases by organophosphorus compounds (Aldridge, 1950) ChE
+ P-X
I
k+l
S ChE.P-X
k-l
k,
2 ChE-P + X
1
SCHEME 1 where ChE is free enzyme, P-X is organophosphoruscompound, ChE. P-X Michaelis complex, and ChE-P is phosphorylated enzyme. The bimolecular velocity constant ( k ; )was estimated following the dilution method of Aldridge (1950). Briefly, acetylcholinesterase was incubated with the inhibitor for various times before tipping the inhibition mixture intoa solution of acetylthiocholine (1 mM) to measure residual activity. Plotting of the In of residual activity ( V;/Vo) against the time for a given concentration is linear, and the slope of the line divided by the inhibitor concentrationgives the k,. This constant was estimated for five different concentrations of each inhibitor. Nucleic Acid Analysis-Cloning and sequencing of the acetylcholinesterase gene from MH19 strain was performed as previously described (Fournier et al., 1989). Briefly, we constructed a genomic library in the EMBL3 X phage vector which was screened with the cDNA clones isolated by Hall and Spierer (1986). Clones of interest were sequenced by the chain terminationmethod (Sanger et al., 1977). Oligonucleotide-directed mutagenesis was performed according t o the Eckstein method (Nakamaye and Eckstein, 1986)using the Spisomer of [a-SIdCTP from Amersham Corp. Analysis of mutant progeny was carried out by hybridization using the mutant oligonucleotide as a probe and by sequencing. Transformation and Genetic Complementation-We used the minigene (pRA)constructed by Hoffmann et al. (1992). A full-length cDNA containing the whole protein-coding sequence was fused to a genomic fragment covering 1.5-kb of promoter sequences. The construct was then inserted in the Carnegie 20 vector and introduced either in r y K o 6 or inAcelZ6/Balancer,rosy- flies by P element-mediated germ line transformation(Rubin and Spradling, 1982). The $" stock carries the wild-type acetylcholinesterase gene, and the Acdz6/ Balancer stock carries a lethal Ace- mutation on one chromosome and a wild-type copy, together with dominant genetic markers, on the homolog balancer chromosome. The Carnegie 20 vector contains the wild type rosy+ gene, an eye-color marker, enabling us to select the flies containing the rosy+-Ace+minigene. Rescues were obtained from crosses between AcelZ6/Balancerflies transformed with the minigene and otherAce-/Balancer mutants flies (AceJ"/MKRS or AceJ5'/ MKRS). Rescued flies were evidenced by the loss of the balancer chromosome recognizable by dominant visible markers.
14271 ~ l y genotype
Ace
-
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Ace -
Ace -
+
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Ace
+
-4.4 -4.5 -4.6
Insecticide toxicity log(x) of Dl50 (mole 1 liter)
-4.7 -4.8 -4.9 -5
-5.1
- 5 '. 23
2 2
4
6
8
10
12
14
16
18
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FIG. 1. Synaptic acetylcholinesterase content and resistance to malathion. Four genotypes were used. pRAAce-/Acerepresents rescued flies. The Ace+ minigene (pRA) is composed of the full-length cDNA and 1.5 kb of the promoter region. Ace'"/MKRS embryos were injected with pRA and transformants were selected for their wild type eye color rosy+. They were then crossed to Ad1'/ MKRSor Df3R126d, and rescued flies ( A ~ e ~ ~ ~ / Aorc eA"d~z 6 / Df3R126d) were selected. In the three lines tested, acetylcholinesterase represents 20-30%of the wild type activity. Ace+/Ace- corresponds to heterozygous strains, AceIz6,Ad", or Ace'". These flies have about 50% activity of the wild type. Ace+/Ace+represents wild type strains, Canton S or 4". pRAAce+/Ace+ represents transformed $06 strains. These flies bear two copies of the normal Ace' gene and the minigene. These strains contain approximately 120130% of the wild type acetylcholinesterase. Sensitivity to malathion was measured in the 12 strains by tarsal contact.
( A ~ e ~ ~ ~ / A c+e iminigene), ~' all and acetylcholinesterase activity is derived from the minigene is located in the central nervous system (Hoffmann et al., 1992). They display only 20-25% of wild type activity (3.1 f 0.3, 3.5 f 0.45, and 2.9 f 0.3 units) probably because we used an intronless minigene insteadof the complete geneand only 1.5 k b of promoter sequences. These flies are also the most susceptible to insecticides (Fig. 1). W e also transformed wild type flies with the minigene which provided an extra gene copy. These flies have about 120-130% of wild type activity (14.8 & 0.8, 15.1 f 0.9, 14.6 f 0.5, and 14.9 f 1 units) and are the most resistant (Fig. 1). These results demonstrate that resistance to organophosphorus compounds is correlated to the amount of acetylcholinesterasein the central nervous system. RESULTS Point Mutation and Resistance to Insecticides-A resistant Amount of Acetylcholinesterase in the Central Nervous Sys- D. melanogaster strain (MH19) was obtained by Morton and tem and Resistance to Insecticides-In Drosophila, acetylchoSingh (1982) froma selection experiment in which increasing linesteraseisencodedbyonegene, Ace. T h ea m o u n t of concentrations of malathion were applied to wild type flies enzyme in a fly, produced by the two copiesof the gene was caught in Ontario (Canada). Originally characterized as aleasily determined by scoring its activity. Activities displayed teredinkineticparametersforacetylcholinehydrolysis, b y the two wildtype strains used, go6 and Canton, were 12.4 MH19 acetylcholinesterase was subsequently shown to be f 1.1and 13.6 f 0.5 units, respectively. Ace deficiency causes resistant to inhibition by malaoxon, the active metabolite of lethality, and many mutations have been isolated (Hall and malathion (Mortonand Holwerda, 1985). The resistant MH19 Kankel, 1976). Strains bearing a lethal Ace mutation can be acetylcholinesterase has the same structure as t h e wild type maintained in a heterozygous state. We tested acetylcholin- enzymeinrespecttomolecularweight,dimericstructure, esterase amounts in three of them: AceIZ6/Ace+,A~e'j'~/Ace+, hydrophobicity or peptidic composition (not shown).To anaand AcelJSo/Ace+. The Ace+ geneis borne by a "balancer lyze the resistance of MH19 acetylcholinesterase to several chromosome" with recessive lethal mutations and aberrations insecticides, we estimated the bimolecular velocity constants preventing crossing over. The activities (6.7 f 0.4, 7.9 0.6, (ki).Comparison witha susceptible strain (Canton S) revealed and 7.9 f 0.7 units) correspond to half of activities found in that MH19 acetylcholinesterase is modified and resistant to wild type flies. Thus, the inactivated allele is not compensated most insecticides (Fig. 2). b y t h e wild type allele carried by the homolog. Accordingly, We have cloned and sequenced the coding regions of t h e than wild MH19 Ace gene. Comparison with the wild these strains are more susceptible to malathion type sequence strains (Fig. 1). We recently succeeded in rescuing Ace- le- (Hall and Spierer, 1986) showed only one non-silent mutation, thality by germ line transformation with a minigene in which namely a T to A substitution resulting in the replacementof the intronless coding region was fused to 1.5-kbof promoter a phenylalanine by a tyrosine at position 368 (Fig. 3A). This
sequences. In rescuedflies
Insecticide Acetylcholinesterase Resistance to and
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phenylalanine residue is conserved in other cholinesterases sequenced so far, suggesting that it is important for catalytic properties.Wehave verified that this base substitution is specific for the MH19 strain and did not originate from a cloning artifact by genomic Southern blot hybridization using as a probe an oligonucleotide bearing the mutation (Fig. 3R). To demonstrate that this change is indeed responsible for the resistance, we introduced the MH19 mutation in the minigene and rescued Ace- flies by P-element transformation.Rescued ki Cantoc ki resistant strain
I
T
flies bearing the mutation are more resistant than theircounterparts transformed with the wild type minigene (Fig. 4A). Analysis of acetylcholinesterase inhibition in two independent rescued lines shows that theminigene bearing the MH19 mutation encodes a resistant enzyme (Fig. 4B),with a lower k, for insecticides. Phenylalanine 368 is therefore involved in the catalytic properties of the Drosophila acetylcholinesterase and mutation to tyrosine produces an enzyme less sensitive to inhibition by insecticides. Interestingly, two phases of inhibitionareapparent in acetylcholinesterase from flies rescued with the MH19 minigene indicating the presence of two proteins, a resistant one and a susceptible one (Fig. 4B).This suggests that theAce'2fi/ Ac&'" combination of two recessive lethal mutationsproduces a small amount of acetylcholinesterase, although not sufficient for viability without the minigene. DISCUSSION
nslaoxon Paraoxon Carbaryl DDVP
DFP
Proporur
FIG. 2. Toxicological response of M H 1 9 acetylcholinestertheresistant ase. Acetylcholinesterasewasaffinitypurifiedfrom strain MH19 and from the susceptihle strain CantonS. The bimolecular velocity constants ( k , )were estimated forfive concentrations for each insecticide. As a measure of resistance, ratios of k, estimated for t h e two strains were calculated.
Amount of Acetylcholinesterase and Resistance to Insecticides-Sensitivity to insecticides is correlated with acetylcholinesterase content. This mechanism of resistance mediated by overexpression has already been described for scavenger or metabolizing proteins. Amplification of esterase genes has been described in mosquitoes (M0uchL.s et al., 1986) and in aphids (Field et al., 1988). Overtranscription of glutathione transferase gene is also involved in a resistant house fly strain (Fournier et al., 1992). Overexpression of target proteins has been described for drug resistance (Schimke, 1982) but not yet for insecticide resistance. We show here that an increase in acetylcholinesterase content in the centralnervous system
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